Enamel demineralization with resin modified gic and conventional composite resin-a comparative in vivo study

Background & Objectives: Fluoride releasing bonding agents can help the orthodontist to minimize enamel demineralization independent of patient cooperation. This in vivo study was conducted to evaluate the effi cacy of resin modifi ed glass ionomer cement (RMGIC) on reducing enamel demineralization around orthodontic brackets and confi rm the superior caries-preventive effect of RMGIC by assessing the mutans streptococci (S. mutans) in plaque samples in vitro. Methods: 60 subjects (aged 14-20 years) scheduled to have premolar extractions as part of the orthodontic treatment plan were selected and randomly divided into 2 groups of 30 each (group 1: the brackets were bonded on the teeth using light cure composite resin and group 2: the brackets were bonded using RMGIC). Plaque scores (modifi cation of plaque index by Silness and Loe) were recorded and plaque samples were collected before bonding, one week and one month after bonding. S.mutans colonies were recorded from the plaque samples inoculated on MSB agar plates, incubated under 95% N2 and 5% CO2 for 48 hours at 370C in a CO2 jar. After 1 month, the right maxillary and mandibular fi rst premolars were debonded, extracted and depth of enamel demineralization area was estimated using polarized light microscope. Results: After statistical analysis, a signifi cantly higher mean depth of demineralized lesions was noticed in group 1 as compared to group 2. A signifi cant difference between occlusal and gingival depth was seen only in group 2, thus illustrating a wedge effect. In group 1, a statistically signifi cant increase in the mean colony forming units (CFU) of S.mutans has been noticed at different time intervals whereas in group2, a signifi cant increase was observed only at 1month. Unlike at 1 month, a statistically signifi cant difference in mean CFU between group 1 and group 2 has been observed at 1 week (P<0.05). Conclusion: Enamel lesions adjacent to the bracket base on teeth bonded with the RMGIC were smaller than those on teeth bonded with a composite resin. The high “burst effect” of fl uoride release for the fi rst few days of RMGIC after bonding is confi rmed by statistically signifi cant reduction in CFU counts of S. mutans in plaque. Research Article Enamel demineralization with resin modifi ed gic and conventional composite resin a comparative in vivo study Gautam G1*, Shashikala Kumari V2, Garima Garg3 and Vikram Shetty4 1MDS, Reader, Department of Orthodontics, YMT Dental College and Hospital, Navi Mumbai, India 2MDS, former Professor and Head, Department of Orthodontics, GDC&RI, Bangalore, India 3MDS, former Lecturer, Department of Periodontics, YMT Dental College and Hospital, Navi Mumbai, India 4MDS, Reader, Department of Orthodontics, YMT Dental College and Hospital, Navi Mumbai, India *Address for Correspondence: Gautam G, Reader, Department of Orthodontics, YMT Dental College and Hospital, Navi Mumbai 410210, India, Tel: +91-9930768280; Email: gautamg_81@yahoo.com Submitted: 20 June 2017 Approved: 28 July 2017 Published: 31 July 2017 Copyright: 2017 Gautam G, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


INTRODUCTION
Enamel decalci ication and white spot lesion (WSL) formation are major clinical problems for patients treated with ixed orthodontic appliances [1]. Results of a meta-analysis by [2]. demonstrated that WSL occurrence is common during ixed orthodontic treatment with an incidence and prevalence rate of 45.8% and 68.4%, respectively, indicating the need for special precautionary steps to be taken to prevent the development of WSLs [1,3], Attributed the WSL (clinically detectable areas of decalci ication), considered to be the precursor of frank enamel caries, to prolonged accumulation and retention of bacterial plaque on the enamel surface adjacent to the orthodontic appliances [4]. The insertion of ixed orthodontic appliances results in a number of new plaque retention sites [5]. Favored sites for accumulation of plaque are around the cervical margins of the teeth, under the bands in areas where the cementing medium has washed out, on the resin surfaces adjacent to bonded attachments, and at the junction of the bonding resin and the etched enamel surfaces [4]. These appliances physically alter the microbial environment with the increased proliferation of facultative bacterial population leading to a marked and localized direct etching of the tooth under the plaque after only one week and visible WSLs within one month adjacent to brackets [4].
Dental plaque is an example of a bio ilm; its presence is natural and it supports the host in its defense against invading microbes. Under certain circumstances, this microbial homeostasis can break down and diseases such as caries can occur. In dental caries, there is a shift toward increased proportions of acid-producing and acid-tolerating species, such as mutans streptococci and Lactobacilli, although other species with relevant traits can participate in demineralization [6,7]. Reported a rise in the number of S. mutans and lactobacilli (LB) after the placement of orthodontic appliances increasing the risk of occurrence of demineralization [8].
Many studies have shown that luoride in trace quantities can increase the degree and rate of remineralization by encouraging and accelerating the regrowth of depleted crystals.15 Fluoride also inhibits the bacterial activity of S. mutans. Fluoride ions interfere with the initial bacterial adhesion and colonization. The inhibitory effect of luoride on bacterial acid production and their growth is enhanced by a decrease in pH of the luoride solution [8]. Fluorides releasing bonding agents have the potential to minimize demineralization around orthodontic brackets independent of patient cooperation [7].
Resin modi ied glass ionomer cements (RMGIC) have been developed that combine the desirable properties of composite resin (bond strength) and glass ionomer ( luoride release and recharge) by modi ication or substitution of the liquid portion of the glass ionomer material [9]. Inhibition of enamel demineralization is shown to occur in vitro to a distance of even 7 mm away from RMGIC restorations [3,10]. The reduction of lesion depth of RMGICs compared to non-luoridated materials ranged from 35% to 75% [10,11].
The purpose of this study is to compare, in vivo, the effect of RMGIC and composite resin for bracket bonding on enamel demineralization adjacent to brackets and con irm with the levels of mutans streptococci [MS] (S.mutans) in plaque.

MATERIAL AND METHOD
After obtaining the institutional ethical approval, sixty subjects (14-20 years) scheduled to have premolar extractions as a part of the orthodontic treatment plan formulated at the Department of Orthodontics and Dentofacial Orthopedics, Government Dental College and Research Institute, Bangalore, satisfying all the inclusion and exclusion criterias were selected for the study. A written informed consent in their respective mother tongue was obtained from the subjects.
3. Right handed subjects (subjects who use their right hand for brushing teeth). 4. Subjects with plaque scores between 0.1 and 0.9 according to the modi ication of plaque index by Silness and Loe.
2. Subjects with irst premolar teeth showing signs of enamel hypoplasia or cracks.
3. Subjects having any chronic disease.
4. Subjects who have received any antibiotics in the previous six weeks.
The subjects were randomly divided into two groups of 30 each using sealed envelope system by an individual not involved in the study.
Group 1: brackets were bonded on the teeth using light cure composite resin (Transbond XT 3M Unitek) Group 2: brackets were bonded on the teeth using RMGIC (Fuji Ortho LC, GC Corp, Tokyo, Japan) With a cheek retractor in position, the buccal enamel was pumiced, washed and dried. A piece of masking tape with a cut-out of the size of a bracket base was applied on the right upper and lower irst premolars. This was done to ensure that there was no etching of the adjacent enamel, which could prematurely initiate demineralization.
For subjects in group 1, brackets were bonded on the teeth with light cure composite resin, Transbond XT (3M Unitek). The enamel was conditioned with 37% phosphoric acid (OrthoSource) for 30 seconds. The brackets were positioned on the buccal surface at the height of contour mesiodistally, in the middle one third occlusogingivally and parallel to the long axis of the tooth and excess was removed with a scaler tip and was light cured for 40-60 seconds.
For subjects in group 2, brackets were bonded on the teeth using RMGIC (Fuji Ortho LC, GC Corp., Tokyo, Japan). The enamel was conditioned with 10% polyacrylic acid (GC Dentin Conditioner) for 30 seconds. The surface was washed for 10 seconds and kept moist as moisture aids in the bonding reaction. Back of the bracket was coated with mixed Fuji Ortho LC. Care was taken not to create any bubbles or voids. The coated bracket was positioned on the tooth and the bracket was irmly pressed against enamel. Excess cement was removed with a scaler tip. Each bracket was light cured for 40-50 seconds.
In both the groups 022΄΄ (0.56mm) MBT TM (3M Unitek) brackets were used for bonding. Brackets were bonded on all the teeth except the left upper and lower irst premolars. Since glass ionomer cement has luoride release potential, molar bands were cemented with zinc phosphate cement. Appliance setup was completed by placing lacebacks with 0.010" steel ligature wires from irst molar to canine and 0.016" Nickel Titanium (NiTi) wire was secured in the bracket slots with conventional elastomeric ligatures (ALASTIK 3M unitek). The archwire was cinched back to prevent anchorage loss in the anterior region.
Precise and similar oral hygiene instructions were given to all the subjects in both the groups. Each subject was given an orthodontic toothbrush and a commercially available sodium luoride dentifrice (1000ppm luoride). Subjects were demonstrated effective plaque removal around the appliance with orthodontic toothbrush. Subjects were also given similar dietary instructions. To ensure compliance, the time of brushing was noted on a checklist by the subjects, which was signed daily by the parent or guardian. The checklist was returned on the next visit.
Plaque scores (modi ication of plaque index by Silness and Loe) were recorded before bonding, one week and one month after bonding and plaque samples were collected from the right upper and lower irst premolars using a sterile hypodermic needle (0.4 mm diameter) The end of the needle was aseptically cut off into a test tube containing 1ml of sterile phosphate buffer solution (pH 7.1).
S.mutans were identi ied on the basis of colony morphology, after incubation of inoculated MSB agar plates under 95% N 2 and 5% CO 2 for 48 hours at 37 0 C. The colony growth was either large rounded colonies having gum drop appearance or darker, small rounded pinpoint colonies. All these colonies were subjected to gram staining to con irm the identi ication as streptococci. Individual colony counts were used as total CFU per sample, transformed to log10 (colony count+1) to normalize the distribution. After 30 days, the brackets bonded on the right upper and lower irst premolars were removed with a sterile debracketing instrument and the teeth were extracted with particular care to avoid damage to the buccal surface of the tooth. The extracted teeth were rinsed carefully with deionized water and stored at 100% relative humidity on wet cotton dampened with 2% formaldehyde in closed container at 400C until analysis. Each tooth was embedded in acrylic and longitudinal buccolingual sections of teeth were made in the middle of the crown with the help of hard tissue microtome (Leica SP 1600) to yield sections 100μm thick. The acrylic slice which now contains embedded tooth section was placed in acetone solution for around 30 minutes thus facilitating the separation of softened acrylic from the tooth section with the help of soft paint brush. After alcohol treatment for 30 minutes, tooth section was placed in a mixture of xylene and alcohol. With the help of a soft paint brush the remaining acrylic debris were cleaned up. In order to ix the tooth section, it was placed in xylene for one hour.
The tooth section was mounted on the glass slide using Dibutyl Pthallate Xylene (DPX). The sections were evaluated with polarized light microscopy (Olympus VX 52). Microphotographs of the gingival half of the buccal surface were taken with ixed magni ication of 20 times. The depth of demineralized lesions was measured using ProGres C3 2.5 image analysis software. The lesion depth in micrometers (μm) for each section was the average of 3 representative measurements (gingival, middle and occlusal) from enamel surface to the depth of the lesion.
The student't' test was used to determine whether there was a statistical difference between Groups in the parameters (plaque score, CFU counts of S.mutans and depth of lesion with enamel demineralization) measured and P value less than 0.05 was taken to be statistically signi icant. The data was analyzed using Statistical Package for Social Science (SPSS, version 13).

RESULTS
A statistically signi icant (P<0.001) increase in mean plaque score was observed within both the groups from baseline (0.49 for group 1 and 0.47 for group 2) to one week (0.60 for group 1 and 0.55 for group 2) and 1 month (1.18 for group 1 and 1.10 for group 2). However, there was no signi icant difference between the groups at different time intervals (P>0.05) (Tables 1,2).
A statistically signi icant (P<0.001), higher mean depth of demineralized lesions was noticed in group 1 (55.65 μm) as compared to group 2 (24.39 μm) ( Table 3, Graph 1). A statistically signi icant difference was observed in mean depth of occlusal, middle and gingival region of demineralized areas in group 1 and group 2 ( Table 4, Graph 2). A signi icant difference between occlusal and gingival depth is seen in group 2 unlike in group 1 ( Table 5, Graph 3).

Mean depth of demineralized area in the two adhesive groups
Graph 1:   A statistically signi icant increase was noticed in the mean CFU at different time intervals in group 1 from baseline (2.30) to 1 week (3.07) and 1 month (4.82). However in group 2, the increase in mean CFU at 1 week (2.54) as compared to that at baseline (2.45) was not found to be statistically signi icant (P>0.05). A statistically signi icant increase was observed at 1 month (4.44) ( Table 6, Graph 4). There was no signi icant difference in the mean CFU between group 1 and group 2 at baseline (P>0.05). There was a higher mean CFU in group1 compared to group 2 at 1 week and 1 month but a statistically signi icant difference was observed only at 1 week (P<0.05) ( Table 7, Graph 5).

DISCUSSION
Fixed appliances are an inseparable part of contemporary orthodontic treatment but a major disadvantage of ixed mechanotherapy is that measurable and signi icant amount of enamel demineralization might occur adjacent to orthodontic bracket even    within one month of bonding. This demineralization is a result of plaque activity in vivo [4]. Fixed appliances physically alter the oral microbial environment so that the proliferation of caries-associated microorganisms, such as S. mutans and LB, is enhanced [8].
As per [12] while examining the effectiveness of a luoride product in preventing dental decay, two aspects should be considered. Firstly, whether the luoride product reduces the number of white spots appearing during treatment and secondly whether it reduces the severity in terms of the size or area of the tooth surface affected or the amount of mineral lost or depth of the decay. When a quantitative method of measuring the amount of mineral lost from enamel or the depth of a carious lesion is used, such as transverse microradiography, polarized microscopy or hardness testing, the tooth being examined has to be extracted and cut into sections. Short experimental periods are inevitable, as delaying the extraction of the tooth will also delay the orthodontic treatment [12].
Signi icantly higher mean depth of demineralized areas noticed in group 1 as compared to group 2 is in accordance with the indings of [2,7,11,13], who emphasized that teeth bonded with the hybrid GIC demonstrated signi icantly smaller enamel lesions adjacent to the bracket base than teeth bonded with a composite resin. Similar indings were also reported by [4,14,15], Using two parallel groups and assessing enamel demineralization by cross sectional microhardness testing [4]. Reported that Fuji Ortho LC reduced enamel demineralization adjacent the brackets by 12% as compared to composite resin (Concise) even though the microhardness of enamel under the brackets bonded with Fuji Ortho LC or Concise was similar, showing that the demineralization was due to caries and not the acid-etching effect of the material [15].
In this study teeth bonded with RMGIC had an approximately 50% reduction in lesion depth compared with those bonded with composite resin, in accordance with the indings of [2,13]. A signi icantly higher mean gingival meauserments (farther from the cement) compared to occlusal measurements (closer to the cement) in group 2 substantiates the wedge effect seen in teeth bonded with RMGIC. Similar wedge effect of RMGIC was also reported by [2,13]. The wedge effect could be due to greater dental plaque accumulation in the gingival/ cervical region of the tooth and the dif iculty in cleaning this area. Protective effect of the luoride released from the RMGIC also decreased outward from the edge of the bracket.
A signi icant difference in the mean plaque score at different time intervals in both the groups was observed, indicating that the placement of ixed appliances in luences the accumulation of plaque [16]. Found that clinical parameters like plaque index, gingival index and bleeding on probing scores had increased signi icantly three months after orthodontic treatment began [16]. The authors did not ind any signi icant difference in the amount of plaque accumulation with the two materials at different time intervals as reported by [7,8].
S. mutans has been strongly associated with caries development in humans [17]. S. mutans is also found on sound tooth surfaces, and its presence does not always indicate the presence of active caries. However, an increased number of these microorganisms on any surface indicate that the disease may be present or may develop in the near future [18,19]. Reported variation in culture results on different medias used for the isolation of S. mutans with the greatest recovery in MSB 10% sucrose medium [19].
A signi icant increase in the mean CFU at different time intervals noticed except between baseline and 1 week in group 2 con irms that the placement of ixed appliances increases the level of S. mutans in plaque and saliva. This inds support with studies of [5,15,18,20]. In group 2 the mean CFU difference between baseline and 1 week was not statistically signi icant unlike group 1, a possible reason being the initial burst of luoride release by Fuji Ortho LC as displayed by [8,21]. This further con irms the overall results of the systematic review by [21], which showed either no difference between the materials, or indicated that RM-GIC has a superior caries-preventive effect [12,22]. Evaluated the antibacterial properties of 4 different orthodontic cements, using both the agar diffusion test and the direct contact test (DCT) and concluded that RMGIC (Fuji Ortho LC) exhibited potent antibacterial activity, which lasted one week and diminished over the next three weeks [12,23]. Showed a good correlation between luoride release and in luence on bacterial growth for RMGIC. However, both effects dropped dramatically over the 180-days period thus con irming the initial burst of luoride release from RMGIC at the time of bonding [23].
On the contrary [24], on the basis of their in vitro study suggested that the amount of luoride released from GIC per se may have little to do with bacterial growth inhibition instead the bacterial inhibitory effect is related to the acid released from the cement as the reduction in GIC pH and the size of bacterial growth inhibition areas were consistently associated. Their study, however, could not rule out the possibility that the antimicrobial activity of GIC could be related both to the initial acid release and luoride levels of the cement samples, since bacterial inhibitory effect of luoride increases as pH decreases [24,25]. In the in vitro study reported that the orthodontic bonding adhesive may release luoride at a rate that affects enamel demineralization rather than bacterial adhesion. Low levels of luoride may be enough to protect enamel against demineralization but may have little effect on inhibiting growth and adhesion of the cariogenic streptococci [26].
Apart from luoride release, other advantages of RMGIC when compared to composite resins for bonding orthodontic brackets as emphasized by [18], like signi icant reduction of chair time, eliminating the need for working in a dry ield and eliminating the need for etching and priming enamel surfaces were also observed during the present study [13].

CONCLUSIONS
1. Teeth bonded with the RMGIC demonstrated signi icantly smaller enamel lesions adjacent to the bracket base than teeth bonded with a composite resin control.
2. In teeth bonded with RMGI cement, depth of enamel lesion increased as the lesion extended farther from the bracket (towards the gingival region of the tooth), illustrating a wedge effect-a decreasing protective effect of the luoride released from the RMGIC in the enamel increasingly distant from the bracket.

3.
A signi icant increase in the number of MS (S. mutans) CFU in plaque was observed within one month after the placement of ixed orthodontic appliances in both the groups.
4. There was a reduction in the CFU counts of S. mutans in plaque around brackets bonded with RMGIC compared with composite resin and this reduction was statistically signi icant at 1 week, emphasizing the high "burst effect" of luoride release for the irst few days of RMGIC after bonding.